Volume 23, Issue 5, Pages 644-652.e5 (May 2018) Complement C3 Drives Autophagy-Dependent Restriction of Cyto-invasive Bacteria  Matthew T. Sorbara, Elisabeth G. Foerster, Jessica Tsalikis, Mena Abdel-Nour, Joseph Mangiapane, Imogen Sirluck-Schroeder, Ivan Tattoli, Rob van Dalen, David E. Isenman, John R. Rohde, Stephen E. Girardin, Dana J. Philpott  Cell Host & Microbe  Volume 23, Issue 5, Pages 644-652.e5 (May 2018) DOI: 10.1016/j.chom.2018.04.008 Copyright © 2018 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2018 23, 644-652. e5DOI: (10. 1016/j. chom. 2018 Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 1 Complement C3 Interacts with ATG16L1 and Increases Autophagy Targeting (A) Left: proteins interacting with ATG16L1 that were identified by Y2H. Right: a schematic of the region of ATG16L1 used as bait in Y2H and of complement C3 identified as the selected interaction domain (green highlights). (B) Flag and C3 blots of immunoprecipitated proteins following a Flag-IP from lysates from cells transfected with Flag-Cherry or Flag-ATG16L1 mixed with C3-negative or C3-positive serum. Representative of n = 3 independent experiments. (C) C3, mouse Ig, and ATG16L1 blots of input and immunoprecipitated proteins following a C3-IP from lysates of cells mixed with purified human C3. Representative of n = 2 independent experiments. (D–G) Autophagy targeting in HCT116 cells 1 hr p.i. with Listeria opsonized with the indicated antibody-depleted serums or PBS. LC3 (D) or ATG16L1 (F) localization is shown in green, C3 staining is shown in red, and DAPI staining is shown in blue. LC3 (E) and ATG16L1 (G) recruitment to Listeria was quantified from n = 3 independent experiments. Full panels are presented in Figure S2. ∗p < 0.05. For (D) and (F), scale bar indicates 5 μm. See also Figures S1 and S2. Cell Host & Microbe 2018 23, 644-652.e5DOI: (10.1016/j.chom.2018.04.008) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 2 Complement C3 Restricts Intracellular Replication of Listeria and AIEC (A) Expression of ATG16L1 and conversion of LC3 to LC3-II in wild-type and ATG16L1−/− HCT116 cells was determined by protein blot (representative of two independent experiments). (B and C) Intracellular levels of Listeria opsonized with C3-positive or C3-negative serum at 1 and 4 hr p.i. in wild-type B) or ATG16L1−/− (C) HCT116 cells. (D) Expression of the 300T or 300A variants of ATG16L1 in ATG16L1−/− HCT116 cells was determined by protein blot (representative of two independent experiments). (E and F) Intracellular levels of Listeria opsonized with C3-positive or C3-negative serum at 1 and 4 hr p.i. in ATG16L1−/− HCT116 rescued with ATG16L1 300T (D) or ATG16L1 300A (E). (G and H) Intracellular levels of AIEC opsonized with C3-positive or C3-negative serum at 1 and 4 hr p.i. in wild-type (G) or ATG16L1−/− (H) HCT116 cells (n = 9). (I and J) Intracellular levels of Shigella opsonized with C3-positive or C3-negative serum at 1 and 4 hr p.i. in wild-type (I) or ATG16L1−/− (J) HCT116 cells. For (B) and (C) and (E)–(J), n = 9–12, indicating wells of cells from 3–4 independent experiments. Input MOIs were adjusted as described in the STAR Methods. ∗p < 0.05, ∗∗p < 0.01. See also Figure S3. Cell Host & Microbe 2018 23, 644-652.e5DOI: (10.1016/j.chom.2018.04.008) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 3 The Omptin Proteases IcsP and PgtE Enable Shigella and Salmonella to Shed C3 and Escape from C3-Dependent Restriction (A) HeLa cells at 1, 2, and 4 hr p.i. with C3-opsonized Shigella. C3 staining is shown in orange, and DAPI staining is shown in blue. (B) Percentage of intracellular Shigella that are C3 positive at 1, 2, and 4 hr p.i. (n = 3). (C) C3 coating of Shigella or Listeria treated with C3-positive serum either immediately following opsonization or after 1 hr incubation in broth culture. (D) The proportional decrease over 1 hr of C3-positive Listeria or Shigella (n = 5). (E) Loss of C3 coating of wild-type, ΔIcsA, or ΔIcsP Shigella treated with C3-positive serum after a 1 hr incubation in broth culture (n = 5 independent experiments). (F) Intracellular levels of ΔIcsP Shigella opsonized with C3-positive or C3-negative serum at 1 and 4 hr p.i. in wild-type or ATG16L1−/− HCT116 cells (n = 15). (G) Intracellular levels of ΔIcsA Shigella opsonized with C3-positive or C3-negative serum at 1 and 4 hr p.i in wild-type or ATG16L1−/− HCT116 cells (n = 15). (H) Loss of C3 coating of wild-type or ΔPgtE Salmonella treated with C3-positive serum after a 1 hr incubation in broth culture (n = 4 independent experiments). (I) Intracellular levels of Salmonella opsonized with C3-positive or C3-negative serum at 1 and 4 hr p.i. in control or ATG16L1 knockdown HCT116 cells (n = 9). (J) Intracellular levels of ΔPgtE Salmonella opsonized with C3-positive or C3-negative serum at 1 and 4 hr p.i. in control or ATG16L1 knockdown HCT116 cells (n = 9). For (F) and (G), input MOIs were adjusted as described in the STAR Methods. For (F), (G), (I), and (J), n indicates wells of cells pooled from 3–5 independent experiments. ∗p < 0.05. Cell Host & Microbe 2018 23, 644-652.e5DOI: (10.1016/j.chom.2018.04.008) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 4 Early Invasion of Listeria into Intestinal Tissues Is Increased in C3−/− Mice (A) C3 protein in the fecal pellets of C3+/+ and C3−/− mice was measured in uninfected mice or 24 and 72 hr p.i. with Listeria. Uninfected: n = 8 C3+/+, 2 C3−/−; 24 hr p.i.: n = 8 C3+/+, 3 C3−/−; 72 hr: n = 4 C3+/+, 2 C3−/−. (B) Listeria CFUs in the gentamycin-protected compartment of the ceacum and colon of C3+/+ and C3−/− littermates were measured at 24 hr p.i. (C) Listeria CFUs in the MLN and spleen of C3+/+ and C3−/− littermates were measured at 24 hr p.i. For (B) and (C): 24 hr: n = 14 C3+/+, n = 14 C3−/−. (D) Listeria CFUs in the gentamycin-protected compartment of the ceacum and colon of ATG7flfl (n = 13) and ATG7fl/fl Vil-Cre (n = 17, 18) littermates were measured at 24 hr p.i. (E) Expression of LC3 and ATG7 mRNAs was quantified in the colons of C3+/+ and C3−/− mice 24 hr p.i. with Listeria (n = 4 C3+/+, n = 4 C3−/−). (F) ATG16L1 protein expression in the colons of C3+/+ and C3−/− 24 hr p.i. with Listeria. (G) LC3-I and LC3-II levels in the colons C3+/+ and C3−/− mice 24 hr p.i. with Listeria. For (E) and (F), data presented are representative of two technical replicates of n = 5 mice per genotype. (H) The LC3-II/LC3-I ratio in the colons of C3+/+ and C3−/− mice. Data presented are the average of two technical replicates from n = 5 mice. ∗p < 0.05, ∗∗p < 0.01. See also Figure S4. Cell Host & Microbe 2018 23, 644-652.e5DOI: (10.1016/j.chom.2018.04.008) Copyright © 2018 Elsevier Inc. Terms and Conditions